This application addresses broad Challenge Area (08) Genomics Topic: 08-DE-103: Epigenomics and Epigenetics of Oral Health and Disease The standard paradigm for characterization of genome wide epigenetic alteration in human cancers has been focused on discovery of specific promoter hypermethylated tumor suppressor genes. We have recently demonstrated that genome-wide epigenetic unmasking and promoter hypomethylation frequently activate candidate proto-oncogenes and tumor specific cancer testes antigens in head and neck squamous cell carcinoma (HNSCC). Past discovery techniques have focused on cell line based pharmacologic demethylating strategies, introducing bias to discovery of epigenetic alterations in tumors. In this project, we integrate direct measurement of genome wide promoter methylation and expression analysis to concurrently define demethylated promoters associated with activated oncogenes, and aberrantly methylated promoters that inactivate tumor suppressor genes in HNSCC. Analysis of patterns of coordinated epigenetic modulation of gene expression will place these alterations within the context of specific biologic pathways. This project will facilitate transition from a limited single gene view, to a pathway based perspective of HNSCC biology and therapeutic development. Hypothesis: Candidate proto-oncogenes and tumor suppressor genes are transcriptionally activated and repressed in association with gene-specific promoter methylation alterations and 1) can be identified using genome wide integrative discovery techniques, and 2) can alter specific biologic pathways in a coordinated fashion. Specific Aim 1: Define a set of potential epigenetically proto-oncogenes and tumor suppressor genes using epigenetic promoter array analysis in HNSCC. These candidates will be integrated with concurrent genome wide expression array analysis and validated by promoter methylation and expression analysis in primary tumors. Rationale: Using pharmacologically demethylated cell line models, we have previously published genome wide discovery approaches to define promoter hypermethylated, inactivated tumor suppressor genes in HNSCC, and recently described an integrative method identifying candidate protooncogenes activated by DNA hypomethylation. These techniques are cumbersome and hampered by cell culture and demethylating drug treatment bias, but have been effective in defining limited sets of altered genes. Use of concurrent genomic promoter methylation and expression arrays in primary HNSCC will allow for direct measurement of these alterations on a genome wide basis without cell culture and drug treatment bias. Identification of significant individual promoter methylation-transcription correlations will identify candidate proto-oncogenes and tumor suppressor genes that are altered by promoter methylation changes. Specific Aim 2: Using concurrent epigenomic and transcriptional analysis of primary HNSCC in Aim 1, we will 1) identify transcriptional targets of methylated regulatory factors, and 2) identify biologic pathways undergoing coordinated changes across individual tumor samples. A selected coordinated, altered pathway will be validated in primary HNSCC. Rationale: We have recently defined the coordinated epigenetic regulation of candidate proto-oncogene activation in HNSCC, via key transcriptional elements. Placement of gene specific epigenetic alteration within the context of genome wide expression alterations will allow us to place epigenetic and transcriptional alterations within the context of common, shared biologic pathways. This will facilitate design of higher impact therapeutic strategies for HNSCC that are pathway specific rather than limited to targeting of single gene alterations. Every year Johns Hopkins Institutions directly generate about $10 billion in economic activity in the State of Maryland, a 43% increase from the $7 billion generated in 2002 and the equivalent of one of every twenty-four dollars in the state's economy today. In 2008, Johns Hopkins Institutions provided 45,000 jobs and created 700 new jobs each year since 2002. Directly and indirectly, Johns Hopkins Institutions support more than 100,000 jobs in Maryland, one of every 29 in the state. In Baltimore City alone Johns Hopkins directly and indirectly supports 60,000 jobs, or 16.7% of all City employment. This application will create or retain four jobs. PUBLIC HEALTH RELEVANCE: The standard paradigm for characterization of genome wide epigenetic alteration in human cancers has been focused on discovery of specific promoter hypermethylated tumor suppressor genes. In this project, we integrate direct measurement of genome wide promoter methylation and expression analysis to concurrently define demethylated promoters associated with activated oncogenes, and aberrantly methylated promoters that inactivate tumor suppressor genes in HNSCC.